The Role of Proline-Rich Protein Tyrosine Kinase 2 in Differentiation-Dependent Signaling in Human Epidermal Keratinocytes  Eva M. Schindler, Magdalena Baumgartner, Erin M. Gribben, Li Li, Tatiana Efimova  Journal of Investigative Dermatology  Volume 127, Issue 5, Pages 1094-1106 (May 2007) DOI: 10.1038/sj.jid.5700662 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Pyk2 is expressed in primary human keratinocytes and localized in the nucleus. (a) Third passage normal human foreskin keratinocytes derived from donors 1 and 2 were cultured in keratinocyte serum-free medium until approximately 80% confluent. Total RNA was then extracted from the cells, and Pyk2 mRNA levels were assessed by RT-PCR. β-Actin levels were monitored as a control to assure equal loading. (b) Total cell extracts were prepared from keratinocytes cultured as described in (a), and assayed for Pyk2 protein expression by immunoblot using anti-Pyk2 antibody. PC12 cell extract was used as a positive control. Equal amounts of protein (40μg per lane) were used in this assay. β-Actin level was assayed to verify equal protein loading. (c) Keratinocytes growing on coverslips were fixed, permeabilized, immunostained with a mouse monoclonal anti-Pyk2 antibody followed by Cy3-conjugated goat anti-mouse IgG, and examined by laser scanning confocal microscopy. Nuclei were stained with Hoechst dye and visualized under a fluorescence microscope. Bar=25μM (d) Keratinocytes were cultured as described in (a). Nuclear (N) and cytosolic (C) fractions were prepared, and assayed for Pyk2 expression by immunoblot using a mouse monoclonal (C-terminal) anti-Pyk2 antibody. (Parallel blots were probed with a rabbit polyclonal (N-terminal) anti-Pyk2 antibody with analogous results, not shown) Successful separation of the nuclear and cytosolic fractions was confirmed by detection of poly(ADP-ribose) polymerase, a nuclear protein, exclusively in the nuclear fraction. PKCδ protein is equally distributed between the nuclear and the cytosolic fractions, ensuring equivalent gel loading. (e) Subnuclear distribution of Pyk2. Images shown in (c and e) are representative of at least five independent experiments using cells isolated from foreskins derived from different donors; data shown in (d) are representative of four independent experiments. Journal of Investigative Dermatology 2007 127, 1094-1106DOI: (10.1038/sj.jid.5700662) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Immunolocalization of Pyk2 in human epidermis. Paraffin-embedded sections of (a) healthy human foreskin or (b) healthy adult human skin were deparaffinized and immunostained with a mouse monoclonal anti-Pyk2 primary antibody followed by Cy3-conjugated goat anti-mouse secondary antibody, and examined by laser scanning confocal microscopy. Nuclei were counterstained with Hoechst dye and visualized under a fluorescence microscope. In (b), the thin arrows in the right panel and the thick arrows in the enlarged image in the right panel, point to the punctuate plasma membrane staining. *Marks nuclei of granular layer cells. (c) Control section with secondary antibody only. Dashed lines, dermal–epidermal junction. Bars=25μM. Images are representative of at least five independent experiments. Journal of Investigative Dermatology 2007 127, 1094-1106DOI: (10.1038/sj.jid.5700662) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Pyk2 is activated by regulators of keratinocyte differentiation. Keratinocytes were cultured as described in Figure 1a, and treated with (a, b, d) 100ng/ml TPA or (e) 1.2mM calcium, as indicated, or (c) infected with empty control adenovirus (EV), or adenoviral vectors encoding PKCη or PKCδ isoforms at a multiplicity of infection=15 for 24hours. (The expression of adenovirus-delivered proteins was confirmed by immunoblot, not shown). In (b and d) keratinocytes were pretreated for 30minutes with or without each of the indicated inhibitors at the following concentrations: (b) 50μM BAPTA-AM, 10μM BIS-IM, 5μM PP2, or 2μM LY 294002; or (d) 10μM Rottlerin. The cells were then treated in the absence or presence of 100ng/ml TPA for (b) 1hour or (d) 30minutes (Inhibitor treatment alone did not influence Pyk2 phosphorylation status, not shown). Total cell extracts were prepared, and Pyk2 activation was assessed by measuring its phosphorylation on tyrosine 402 (Y402), a major autophosphorylation site, using phosphotyrosine 402-specific antibody (pY402-Pyk2). Total Pyk2 level was monitored using anti-Pyk2 antibody. β-Actin level was assayed to assure equal protein loading. The data presented in each panel are representative of at least three independent experiments. Journal of Investigative Dermatology 2007 127, 1094-1106DOI: (10.1038/sj.jid.5700662) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Treatment with differentiating agents does not affect subcellular localization of endogenous Pyk2 protein. (a) Keratinocytes growing on coverslips were cultured in culture medium with 0.03mM calcium (Control), or treated with 1.2mM calcium or 100ng/ml TPA for the indicated periods of time, fixed, permeabilized, and immunostained with a monoclonal mouse anti-Pyk2 antibody followed by Cy3-conjugated goat anti-mouse IgG, and examined by laser scanning confocal microscopy. Bars=25μM. (b) Cultured human keratinocytes were treated without (untreated), or with 100ng/ml TPA, or 1.2mM calcium. The cells were harvested and nuclear extracts were prepared at the time points indicated. The protein levels of Pyk2 were assessed by immunoblot using anti-Pyk2 antibody. Equal protein loading was verified by membrane stripping and reprobing with antibody specific to nuclear marker proteins poly(ADP-ribose) polymerase and Lamin A/C (not shown). Data presented in each panel are representative of three independent experiments. Journal of Investigative Dermatology 2007 127, 1094-1106DOI: (10.1038/sj.jid.5700662) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Immunolocalization of pY402-Pyk2 in keratinocytes. Cells growing on coverslips were cultured in culture medium with 0.03mM calcium (Control), or treated with medium containing 1.2mM calcium (Calcium), or 100ng/ml TPA (TPA) for 2hours, fixed, permeabilized, and immunostained with rabbit polyclonal anti-pY402-Pyk2 antibody followed by Alexa Fluor-conjugated goat anti-rabbit IgG, and examined by laser scanning confocal microscopy. No staining was observed when normal rabbit IgG was used in place of primary antibody, confirming the specificity of signal (not shown). Bar=25μM. Data shown are representative of three independent experiments. Journal of Investigative Dermatology 2007 127, 1094-1106DOI: (10.1038/sj.jid.5700662) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Pyk2 increases hINV promoter activity. (a) Cultured human keratinocytes were transfected with the full-length hINV promoter construct that includes 2,473bp of the hINV promoter region linked to the luciferase reporter gene in pGL2-Basic plasmid, and empty control expression vector or plasmid encoding FLAG epitope-tagged wild-type Pyk2 (Pyk2wt). After 48hours, the cells were treated with culture medium in the absence or presence of 50ng/ml TPA for an additional 24hours. The cells were then harvested and lysates were assayed for luciferase activity. Luciferase activity was normalized per (μg) of protein. The expression of vector-produced Pyk2 was confirmed by immunoblot using anti-FLAG antibody (inset). Data shown are mean±SE of five independent experiments, each performed in triplicate. RLU – relative luciferase units. (b) Differential effects of PKC inhibitor BIS-IM on TPA- and Pyk2-dependent hINV promoter activity. Cultured human keratinocytes were transfected with pINV-2473 reporter plasmid and empty control expression vector (vector) or plasmid encoding wild-type Pyk2 (Pyk2wt). 24hours post-transfection, the cells were treated in the presence or the absence of 5μM BIS-IM for additional 24hours. Parallel vector-transfected dishes were treated for 24hours with or without 50ng/ml TPA (vector+TPA). BIS-IM was added 30minutes before addition of TPA. Cell extracts were assayed for luciferase activity. Data shown are mean±SE of three independent experiments, each performed in triplicate. Journal of Investigative Dermatology 2007 127, 1094-1106DOI: (10.1038/sj.jid.5700662) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Pyk2 increases endogenous hINV levels. (a) Keratinocytes were infected with control GFP-encoding adenovirus (Ad-GFP), or adenovirus encoding GFP-tagged Pyk2 (Ad-GFP-Pyk2) at a multiplicity of infection of 30. After 48hours, the cells were harvested, total cell extracts were prepared, and expression of adenovirus-delivered Pyk2 was confirmed by immunoblot using anti-Pyk2 antibody and anti-GFP antibody. β-Actin levels were monitored to assure equal loading. The phosphorylation status of Pyk2 was monitored using phospho-tyrosine 402-specific antibody (pY402-Pyk2). (b) Keratinocytes were infected with the indicated adenovirus as described in (a). At 24hours post-infection, the cells treated in the absence or presence of 50ng/ml TPA for an additional 24hours, total cell extracts were then prepared, and involucrin protein expression was assessed by immunoblot. β-Actin was used as a loading control. Data shown are representative of five independent experiments performed using cells derived from different donors. (c) Pyk2 induces tyrosine phosphorylation of Src in keratinocytes. Cells were infected with the indicated adenovirus as detailed in (a). Total cell extracts were prepared 48hours post-infection, and the levels of phosphorylation of Src on tyrosine 416, which is indicative of Src enzymatic activity, was assessed by immunoblot using phosphotyrosine 416-specific antibody (pY416-Src). Total Src levels were measured using anti-Src antibody. Data shown are representative of at least three independent experiments. Journal of Investigative Dermatology 2007 127, 1094-1106DOI: (10.1038/sj.jid.5700662) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Pyk2 increases levels of AP1 transcription factors Fra-1 and JunD. Keratinocytes were infected with control GFP-encoding adenovirus (Ad-GFP), or adenovirus encoding GFP-tagged Pyk2 (Ad-GFP-Pyk2) at a multiplicity of infection of 30. After 24hours the cells were treated in the absence or presence of 50ng/ml TPA for an additional 24hours. Nuclear extracts were then prepared and AP1 factor expression was monitored by immunoblot using an appropriate AP-1 factor-specific antibody. p38δ Level was assayed to assure equal protein loading, as a substantial quantity of the endogenous p38δ protein have been shown to reside in the nucleus in keratinocytes (Efimova et al., 2004). Journal of Investigative Dermatology 2007 127, 1094-1106DOI: (10.1038/sj.jid.5700662) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions